Hematopoiesis is a regulated developmental cascade that provides an accessible model system in mammals to study the development of distinct lineages. However, the regulatory pathways that control hematopoiesis are as yet poorly understood. The proto-oncogene PU.1/Spi-1 was originally cloned as the of site of retroviral integration in Friend virus induced erythroleukemias. Deregulation of PU.1 has subsequently been shown to be sufficient for erythroblast transformation. PU.1 has also been suggested to be a regulator of hematopoiesis based on its restricted expression pattern (primarily in B-cells and monocytes). To better understand the function of PU.1 during hematopoiesis, the gene was targeted via homologous recombination in embryonic stem (ES) cells and "knock-out" mice were generated. Analysis of the hematopoietic system revealed that lymphoid and myeloid development was completely abrogated in mutant embryos (Scott et al., 1994). In vitro clonogenic and radioprotection assays with PU.1-/- hematopoietic cells have demonstrated that the defect in myeloid and lymphoid development is cell intrinsic. In addition, the myeloid differentiative capacity of PU.1 -/- ES cells can be rescued with a transgene which expresses PU.l under the control of its own promoter. The ability to "rescue" PU.1-/- ES cells allows the determination of which functional domains of the PU.1 protein and which cis-regulatory promoter elements are required for myeloid and lymphoid differentiation. The role of PU.1 in the later stages of myeloid and lymphoid development also remains to be determined. In order to further our understanding of the role that PU.1 plays during hematopoiesis, we propose the following Specific Aims: Aim I. To establish in vivo the functional domains of the PU.1 protein required for myeloid and lymphoid development. Aim II. To establish in vivo the PU.1 regulatory elements necessary for myeloid and lymphoid development. Aim III. Determine the role of PU.1 in the later stages of lymphoid and myeloid development.